Engineered Production of Isoprene from the Model Green Microalga Chlamydomonas Reinhardtii

Abstract
Isoprene (C5H8) is the volatile hemiterpene monomer of the natural polymer rubber and has been recognized as feedstock for the chemical industry. Isoprene is naturally produced by a wide range of species via the methyl erythritol 4-phosphate (MEP) isoprenoid biosynthesis pathway. In the algal chloroplast, sunlight energy is used in the conversion of CO2 into G3P and pyruvate for cellular carbon. These 3-carbon compounds are used by the MEP pathway to make the 5-carbon isopentyl- and dimethylallyl pyrophosphate (IPP and DMAPP, respectively) precursors of carotenoid pigments and cellular sterols. Isoprene synthases (IspSs) produce isoprene by cleavage of the pyrophosphate group of DMAPP to yield molecular isoprene. In this work, we benchmarked expression of four IspSs from different plant species in a high-density cultivation enabled strain of model green microalga Chlamydomonas reinhardtii (UPN22): Ipomoea batatas (IbIspS), Eucalyptus globulus (EgIspS), Pueraria montana (PmIspS) and Populus alba (PaIspS). Of the tested synthases, IbIspS yielded the highest and most stable isoprene yields in alga. To further enhance isoprene production, up- and downstream portions of the MEP and carotenoid pathways were perturbed to investigate their in uence on isoprene yields. The 1- deoxyxylulose 5-phosphate synthase from Salvia pomifera (SpDXS) and isopentenyl diphosphate isomerase from Saccharomyces cerevisiae (ScIDI) were overexpressed to modify upstream ux to DMAPP. Carotenoid biosynthesis was perturbed through overexpression of the Daucus carota lycopene beta cyclase (DcLCYB) and the C. reinhardtii beta carotene ketolase (CrBKT). A synergistic effect of CrBKT and ScIDI co-expression with IbIspS was found to result in robust isoprene generation from the eukaryotic alga. Using 20-photobioreactors, the interplay of light and temperature was used to determine that culture temperature, rather than incident light, had the greatest effect on isoprene yields from the algal host.

Citation
Yahya, R., Wellman, G., Overmans, S., & Lauersen, K. (2023). Engineered Production of Isoprene from the Model Green Microalga Chlamydomonas Reinhardtii. KAUST Research Repository. https://doi.org/10.25781/KAUST-Y0SZ3

Conference/Event Name
Metabolic Engineering conference (ME15)

DOI
10.25781/KAUST-Y0SZ3

Additional Links
https://www.aiche.org/imes/conferences/metabolic-engineering-conference/2023/me15-poster-assignments

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